[Interaction of phosphorylase B with SH-reagents and properties of the modified enzyme].
The interaction of phosphorylase B with the SH-reagents, i.e. 2-chloromercuri-4-nitrophenol and ethylmercurichloride was studied. It was shown that phosphorylase B inhibition obeys the pseudo-first-order kinetics, the inactivation rate constants being equal to 11 M-1 s-1 and 17,5 M-1 s-1, respectively. Data from the SH-group titration with 2-chloromercuri-4-nitrophenol and p-chloromercuri benzoate suggest that the number of modified cysteine residues and the amount of bound 2-chloromercuri-4-nitrophenol in the phosphorylase B dimer is equal to 2. In the modified phosphorylase B the absorption maximum of pyridoxal phosphate is decreased at 330 nm and is increased at 410 nm. The binding of 2-chloromercuri-4-nitrophenol is accompanied by quenching of the protein and coenzyme fluorescence. Upon interaction with ethylmercurichloride only the pyridoxalphosphate fluorescence is quenched. The increase of the spin label mobility in the modified enzyme calculated from the EPR spectra of the spin-labelled preparations is indicative of the changes in the protein conformation coupled with the blocking of one SH-group in the enzyme monomer. The rate of enzyme inactivation under effects of the SH-reagents is a function of pH and is considerably increased within the pH range of 5.7-6.7. The pH-optimum of activity of partly modified enzyme remains practically unchanged; however, at the pH shift towards the acidic values the activity is drastically decreased as compared to that of the native enzyme. The data obtained suggest that the enzyme inactivation is due to modification of one SH-group in the phosphorylase B monomer vicinal to the pyridoxal phosphate binding site and probably involved in the enzymatic reaction.
D000818:Animals; D007700:Kinetics; D046911:Macromolecular Substances; D010763:Phosphorylase b / Q000378:metabolism*; D006005:Phosphorylases / Q000378:metabolism*; D011732:Pyridoxal Phosphate / Q000032:analysis; D011817:Rabbits; D013053:Spectrophotometry; D013439:Sulfhydryl Reagents / Q000494:pharmacology*
D046911:Macromolecular Substances; D013439:Sulfhydryl Reagents; D011732:Pyridoxal Phosphate; D010763:Phosphorylase b; D006005:Phosphorylases
Vzaimodeĭstvie fosforilazy v C SH-PEAgentami i izuchenie svoĭstv modifitsirovannogo fermenta.